Fig 2: Insulin inhibits isoproterenol-stimulated lipolysis in 3T3-L1 adipocytes via the AKT/PKA/HSL pathway.We treated differentiated adipocytes with 100 nM insulin and 1 μM isoproterenoland examined the effects on lipolysis(in triplicate). (a)Western blot analyses ofAKT, PDE3B, PKA, HSL and ATGL protein levels in the insulin, isoproterenol and control groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (b)The phosphorylated protein/total protein ratios of AKT, PKA and HSL were calculated, and the control ratio was normalized to 1. (c)Western blot analyses of PDE3B protein levels in the insulin, isoproterenol and control groups. Differentiated 3T3-L1 adipocytes were treated with different doses of insulin (1, 10,100 nM) and 1 μM isoproterenol for 24 h(in triplicate). (d) Cellular triglycerides were stained with Oil red O. Bar, 50 μm. (e) The amount of lipids was quantified by the Oil red O staining technique. (f)The concentration of glycerol in the medium was detected in different groups(g, i)Western blot analyses of AKT, PDE3B, PKA, HSL and ATGL protein levels in different groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (h) The phosphorylated protein/total protein ratios were calculated, and the control ratio was normalized to 1.(j)cAMP levels in different groups. (k) The expression of PDE3Bwas determined in different groups by Western blot.

Fig 3: The effect of BNG-1 on (A) Expressions of hepatic IL-1β, TNF-α, PDE3B, and adipose tissue PDE3B, and (B) hepatic and (C) adipose cAMP levels in mice. 1Bars are means ± SD. 2Con (8)+saline = mice received standard control diet for 8 weeks; HFD (8)+saline, HFD (8)+LBNG, HFD (8)+MBNG, and HFD (8)+HBNG groups = mice received high fat diet for 8 weeks and supplemented daily with 0, 0.026, 0.035, and 0.052 g BNG-1/30 g body weight at the last 4 weeks, respectively. 2PDE3B = phosphor-diesterase 3B; IL-1β = interleukin-1β; TNF-α = tumor necrosis factor-α.

Fig 4: The effects of the depletion of AKT or PKA on the regulation of lipolysis by insulin.Differentiated adipocytes were transfected with AKT siRNAortreated with the PKA inhibitor and incubated in the presence of 100 nM insulin and 1 μM isoproterenol for 24 h (in triplicate). (a-b)Protein expression of PDE3B, PKA, HSL and ATGL in 3T3-L1 adipocytes transfected with AKT siRNA. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (c-d)Relative protein expression of PDE3B and the phosphorylated protein/total protein ratios for PKA, HSL, and ATGL; the control ratio was normalized to 1. (e)Determination of cAMP levels. (f) Glycerol released into the medium after transfection with an siRNA targeting AKT. (g)Western blot analyses of HSL and phosphorylated HSL (p-HSL) protein expression in 3T3-L1 adipocytes treated with different doses of the PKA inhibitor. (h) The phosphorylated protein/total protein ratio for HSL in 3T3-L1 adipocytes treated with different doses of the PKA inhibitor. (i) Glycerol released into the medium after treatment with different doses of the PKA inhibitor. *P<0.05; **P<0.01.

Fig 5: Effects of PID1 expression on lipolysis and the phosphorylation of AKT/PDE3B/PKA/HSL signaling molecules and ATGL.Preadipocytes were subjected to PID1knockout or upregulation and allowed to differentiate into 3T3-L1 adipocytes; these cells were treated with 1 μM isoproterenol and 100 nM insulin for 24 h(in triplicate). (a-b)Immunofluorescence analysis was performed to assess the expression of the PID1 gene in empty vector cells, PID1-overexpressing cells, and control cells.(c)RT-PCR analyses of the mRNA expression of PID1 in empty vector cells, PID1-overexpressing cells, and control cells. (d)Glycerol released into the medium after the upregulation of PID1.(e-f)Protein expression of AKT, PDE3B, PKA, HSL and ATGL in empty vector cells, PID1-overexpressing cells, and control cells. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control.(g)The phosphorylated protein/total protein ratios for AKT, PKA, HSL, and ATGL in 3T3-L1 adipocytes after transfection with the PID1 overexpression plasmid. (h)The expression of PDE3B was determined by Western blot after the PID1 overexpression plasmid. (i-j)Protein expression of AKT, PDE3B, PKA, HSL and ATGL after transfection with PID1 shRNA. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (k)RT-PCR analyses of mRNA after transfection with PID1 shRNA. (l) Glycerol was released into the medium after knockdown of PID1. (m)Phosphorylated protein/total proteinratios for AKT, PKA, HSL, and ATGL after PID1 knockdown. (n)The expression of PDE3B was determined by Western blot after transfection with PID1 shRNA.